The innate immune system produces reactive oxygen species and reactive nitrogen intermediates. Therefore, resistance to oxidative and nitrosative stress is important for pathogens that encounter macrophages and neutrophils. Data from our previous granting period has indicated that the thioredoxin system i) is induced during oxidative and nitrosative stress, ii) is essential for resistance to oxidative and nitrosative stress, and iii) is important for the virulence of Cryptococcus neoformans. There are three interrelated, yet independent, specific aims in this application that will further define the involvement of the thioredoxin system in the complex mechanisms of resistance to reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI). In the first Aim, we propose to localize the components of the thioredoxin system in C. neoformans. Our preliminary data suggests that a thiol specific antioxidant protein (Tsa1) is localized to the periphery of the cell. Our hypothesis is that C. neoformans (and potentially other fungi) has co-opted a unbiquitous, conserved antioxidant system that is used by other organisms to counteract internally derived ROS, and has localized it to the periphery of the cell, where it is effective for combating external ROS. Given that the thioredoxins have been thought to be critical for the recycling of the thiol peroxidase and that Tsa1 is localized in the cell periphery, the precise localization (cell wall, cell membrane, membrane associated, or mitochondrial) of the thiol peroxidases and thioredoxin and the potential for trafficking between the outer periphery and the cytoplasm is important to understand. In this aim we will localize Tsa1, Trx1, and Trx2 more specifically by co-localization studies with known proteins and determine the signals that are important for localization. In the second Aim, we will determine the post-translational modifications that occur to the thiol peroxidase during treatment with nitric oxide or peroxide, and determine if these are critical for function. We will also determine the mechanism by which Tsa1 is recycled. In the third aim, we will determine the mechanism of transcriptional regulation of the thioredoxin system in response to nitrosative stress. We have preliminary data that we have identified a unique transcription factor that in partially responsible for responses to nitric oxide. We will search for an additional transcription factor that mediate the induction of TSA1 by nitric oxide and will determine the mechanism by which a nitric oxide responsive transcription factor is activated.